The Role Of Extracellular Calmodulin On Calcium Signals In Pollen Tube Of Self-Incompatibility In Pyrus Pyrifolia Nakai

Using Pyrus pyrifolia cultivars'Hosui'(S3S5) and'Imamuraaki'(S1S6) as materials, the effects of calmodulin protein (CaM) on pollen germination, tube growth and the dynamic of cytosolic free calcium concentration ([Ca2+]i) in vivo and in vitro were studied and the signal transduction mechanisms of stylar S-RNase and G protein regulators mediating the pollen tube growth were discussed. The main results as follows:1. In the self-incompatibility, CaM could increase pollen germination and pollen tube growth and relieves S-RNase inhibition on the concentration of Ca2+ through improving [Ca2+]i.It also could change the pollen tube tip [Ca2+]i and [Ca2+]i shaking and induce specific signaling of "calcium transient" in pollen. The Ca2+ channel was activated by adding extra-CaM, which could revive the inhibition of Self-S-RNase on Ca2+ channel activity.2. The inhibitor of G protein PTX could eliminate the promotion of CaM on pollen germination and tube growth. The activation of G protein CTX could reverse the suppression of anti-CaM serum on pollen germination and tube growth. The CTX and PTX mediated [Ca2+]i raise and decrease in pollen and pollen tube, respectively. CTX could reverse the suppression of anti-CaM serum on pollen tube [Ca2+]i, and PTX could keep down the promotion of CaM on pollen tube [Ca2+]i. This show that G protein was on the down stream of CaM.3. The result showed CaM cooperate with G protein can release the incompatibility S-RNase restain process and they can delay the [Ca2+]i downtrend. They also made the Ca2+ of tube tip and whole pollen tube increasing, Ca2+grads disappearing and changed the [Ca2+]i shaking. There was no effect of CaM cooperate with G protein on the pollen deal with compatibility S-Rnase. These indicated that CaM cooperated with G protein could participate in self-incompatibility of pear and play a role on the downstream of S-Rnase. So we could presume a signal route of style S-RNase-extra-CaM-G protein.4. Using the immunity colloidal gold electron microscopy, CaM was found to be localized near by the plasma membrane. In addition, there was some CaM on cell wall. CaM was far from plasma membrane in incompatible pollen and moved to the middle-part of pollen tube by S-RNase treatment 24h late. There was no in evidence variation of CaM localization in compatible pollen tube.5. The variation of style auto-fluorescence after exogenous calmodulin and anti-CaM serum treatment on self-and cross-pollination were studied with laser scanning confocal microscope (LSCM). The results showed that after pollination 12h CaM treatment on cross-pollination made the auto-fluorescence.of topside style reduce and the tail of style increase, but treatment on self-pollination made whole style auto-fluorescence increase. Anti-CaM serum treatment on cross-pollination increase the whole auto-fluorescence and reatment on self-pollination changed the distributing disciplinarian of style auto-fluorescence. After pollination 72h, self-pollination style auto-fluorescence acutely changed. CaM treatment on cross-pollination style auto-fluorescence increased and the highest value was at ovary. CaM treatment on self-pollination and anti-CaM serum treatment on self-and cross-pollination changed the distributing disciplinarian of auto-fluorescence.6. The variation of style Ca2+ location and structural after CaM and anti-CaM serum treatment on self-and cross-pollination were studied in vivo by electron microscopy. The results showed that there was no calcium grains in non-pollination and cross--pollination style. The self-pollination style intercellular spaces Ca2+ grains disappeared with the vacuole augment and intercellular spaces decrease after CaM treatment. The self-pollination style intercellular spaces Ca2+ concentration increased and intercellular spaces enlarged after anti-CaM serum treatment.7. Transmission electron microscopy was adopted study the effects of the ultrastructures of self-and cross-pollination pollens and style.The results showed that in vivo pollination 3h, ultrastructures of compatible and incompatible style was same. It was full of endoplasmic reticulum, mitochondrial and vacuole in pollen tube. After pollination 24h, the configuration was integrity. The endoplasmic reticulum distributed in front of pollen tube. Mitochondrial reduced and vacuole was large. The cell wall was thick in the middle of pollen tube. After self pollination 48h, cell membrane of pollen tube tip ruptured and disrepaired. There was no organelle in pollen tube.